Project 1: The Interaction of DA/Glutamate Signaling in Vulnerability to Aberrant Alcohol Seeking Jane Taylor, Ph.D. Abstract Although many people can casually consume alcohol, some people develop alcohol use disorder (AUD), especially those that are family history positive (FHP) for AUD. In AUD, drinking becomes habitual and alcohol cues become powerful triggers that invigorate alcohol seeking and ultimately trigger relapse to alcohol consumption. Unfortunately, there are few effective therapies that reduce the strength of these cues. Our goal is to use cutting edge behavioral, transgenic and viral techniques to provide a more complete understanding of the neurobiological mechanisms of habitual alcohol seeking and relapse. We will test the overarching hypothesis that habitual alcohol seeking and relapse in FHP and AUD is due to enhanced D1-R-mediated NMDA-R function and decreased D2-R-mediated NMDA-R function in the dorsal striatum. In CTNA3 we found that vulnerability to habitual ethanol (EtOH) seeking and relapse in mice was associated with two distinct endophenotypes of enhanced cue reactivity. First, mice that exhibited high Pavlovian Approach (PA) exhibited greater habitual alcohol seeking relative to mice that exhibited low PA. Second, mice that exhibited high Pavlovian to Instrumental Transfer (PIT) exhibited greater cue-induced reinstatement of EtOH seeking relative to mice that exhibited low PIT. Here, we will study the molecular basis of these predictors of compulsive EtOH seeking. Specifically, we propose to investigate the role of DA signaling cascades that underlie the transition from goal-directed to habitual alcohol seeking (Aim 1), and cue-induced relapse (Aim 2). We will use cell- specific viral knockdown of the key DA-signaling molecules that regulate NMDA-R function, the tyrosine kinase Fyn and the tyrosine phosphatase STEP, in D1-Cre and D2/A2A-Cre mice to selectively determine the roles of Fyn and STEP in D1 or D2 dorso-medial and dorso-lateral striatal neurons: The AAV-flox-Fyn-shRNA and AAV-flox-STEP-shRNA constructs were designed specifically for this project. We will then test a novel systemic Fyn inhibitor to prevent the transition to habitual EtOH-seeking. In CTNA3, we also found that cortical neural cell adhesion molecule (NCAM) expression predicted cue-reactivity and was required for cessation of EtOH-seeking behaviors. The Clinical Core identified NCAM-1 as the gene most closely associated with Fyn kinase in the AUD ?risk? gene network. We will investigate dorso-striatal NCAM signaling-dependent NMDA-R expression in the regulation of habitual alcohol seeking and relapse through enhanced polysialation: The AAV- PST virus is a novel means to enhance PSA-NCAM expression. Finally, we are uniquely positioned to study sex differences in EtOH seeking and DA/NMDA signaling. In CTNA3 we found the transition to habitual EtOH seeking is accelerated in male mice relative to female mice, and this depends on chromosomal complement rather than gonadal status: Cre-mouse lines will be used for the first time to extend these findings. Overall, this project will yield reverse translational data through parallel behavioral assessments and targeted brain-specific molecular analyses that will inform the use of Fyn-kinase inhibition as a potential pharmacotherapy for AUD.